GABA-B2 receptor

ABSTRACT

The invention provides isolated polynucleotide molecules encoding a novel neuropeptide GABA-B receptor (designated GABA-B). These isolated polynucleotide molecules can be used to express the receptor in cells which can then be used to screen compounds for agonist and antagonist activity.

FIELD OF THE INVENTION

The present invention relates to isolated polynucleotide molecules whichencode a novel transmembrane G-protein coupled receptor designatedGABA-B2. The novel receptor appears to be activated by theneurotransmitter y-amino butyric acid (GABA).

BACKGROUND OF THE INVENTION

γ-amino butyric acid (GABA) is the principal inhibitory neurotransmitterin the brain, whose action is mediated by two types of receptors, GABA-Aand GABA-B. GABAergic inhibitory neurons typically form short pathways(e.g. from striatum to substantia nigra and from cerebellar cortex todeep cerebellar nuclei), although at least one long pathway projectingfrom the posterior hypothalamus to the cerebral cortex has also beenrecognized. This long pathway is believed to provide a direct pathway bywhich limbic, emotional and visceral information may be transferred tothe cortex (Vincent et al., Science 220: 1309-1311, 1993).

GABA-B receptors, such as the GABA-B1a and GABA-B1b receptors, arepredominantly present in the brain where they are believed to play amajor role in learning and memory. In view of these functions and theknown benefit of GABA-B agonists (e.g. baclofen) in the treatment ofspasticity, anxiety and depression, there is considerable interest inisolating genes encoding GABA-B receptor subtypes so as to, enable therecombinant production of GABA-B receptors for the development of noveltherapeutics.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides an isolatedpolyniucleotide molecule encoding a GABA-B2 receptor or a functionallyequivalent fragment thereof.

Preferably, the encoded GABA-B2 receptor is characterised by theN-terminal amino acid sequence: MASPRSSGQP (SEQ ID NO: 1)

More preferably, the isolated polynucleotide molecule encodes a humanGABA-B2 receptor of about 941 amino acids.

Most preferably, the isolated polynucleotide molecule encodes a GABA-B2receptor having an amino acid sequence substantially corresponding tothat shown as SEQ ID NO: 2.

The polynucleotide molecule of the first aspect may comprise anucleotide sequence substantially corresponding to, or showing at least90% (more preferably, at least 95%) homology to that shown atnucleotides 1 to 3256 or nucleotides 140 to 2962 of SEQ ID NO: 3 or anyportion thereof encoding a functionally equivalent GABA-B2 receptorfragment.

The isolated polynucleotide molecule may be incorporated into plasmidsor expression vectors (including viral vectors), which may then beintroduced into suitable bacterial, yeast, insect and mammalian hostcells. Such host cells may be used to express the GABA-B2 receptorencoded by the isolated polynucleotide molecule.

Accordingly, in a second aspect, the present invention provides amammalian, insect, yeast or bacterial host cell transformed with thepolynucleotide molecule of the first aspect.

In a third aspect, the present invention provides a method of producingGABA-B2 receptors or functionally equivalent fragments thereof,comprising culturing the host cell of the second aspect under conditionsenabling the expression of GABA-B2 receptors or functionally equivalentfragments thereof.

Preferably, the host cell is mammalian or of insect origin. Where thecell is mammalian, it is presently preferred that it be a Chinesehamster ovary (CHO) cell, monkey kidney (COS) cell or human embryonickidney 293 cell. Where the cell is of insect origin, it is presentlypreferred that it be an insect Sf9 cell.

In a preferred embodiment, the GABA-B2 receptors or fragments thereofare expressed onto the surface of the host cell.

By using the polynucleotide molecule of the present invention it ispossible to obtain GABA-B2 receptor protein or fragments thereof in asubstantially pure form.

Accordingly, in a fourth aspect, the present invention provides aGABA-B2 receptor or a functionally equivalent fragment of said receptor,in a substantially pure form.

In a fifth aspect, the present invention provides an antibody capable ofspecifically binding to the GABA-B2 receptor of the fourth aspect. Suchantibodies may be produced by any of the methods routine to the art.

In a sixth aspect, the present invention provides a non-human animaltransformed with a polyniucleotide molecule according to the firstaspect of the present invention.

In a seventh aspect, the present invention provides a method fordetecting agonist or antagonist agents of a GABA-B2 receptor, comprisingcontacting a GABA-B2 receptor, functionally equivalent fragment thereofor a cell transfected with and expressing the polynucleotide molecule ofthe first aspect, with a test agent under conditions enabling theactivation of a GABA-B2 receptor, and detecting an increase or decreasein activity of the GABA-B2 receptor or functionally equivalent fragmentthereof.

An increase or decrease in activity of the receptor or functionallyequivalent fragment thereof may be detected by measuring changes in cAMPproduction, Ca²⁺ levels or IP3 turnover after activating the receptormolecule with specific agonists or antagonists.

In a further aspect, the present invention provides an oligonucleotideor polynucleotide probe comprising a nucleotide sequence of 10 or morenucleotides, the probe comprising a nucleotide sequence such that theprobe specifically hybridises to the polynucleotide molecule of thefirst aspect under high stringency conditions (Samibrook et al.,Molecular cloning: a laboratory manual, Second Edition, Cold SpringHarbor Laboratory Press).

In a still further aspect, the present invention provides an antisenseoligonucleotide or polynucleotide molecule comprising a nucleotidesequence capable of specifically hybridising to an mRNA molecule whichencodes a GABA-B2 receptor so as to prevent translation of the mRNAmolecule.

Such antisense oligonucleotide or polynucleotide molecules may include aribozyme region to catalytically inactivate mRNA to which it ishybridised.

The polynucleotide molecule of the first aspect of the invention may bea dominant negative mutant which encodes a gene product causing analtered phenotype by, for example, reducing or eliminating the activityof endogenous GABA-B2 receptors.

The term “substantially corresponding” as used herein in relation toamino acid sequences is intended to encompass minor variations in theamino acid sequences which do not result in a decrease in biologicalactivity of the GABA-B2 receptor. These variations may includeconservative amino acid substitutions. The substitutions envisaged are:

G, A, V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P,Nα-alkalamiino acids.

The term “substantially corresponding” as used herein in relation tonucleotide sequences is intended to encompass minor variations in thenucleotide sequences which due to degeneracy in the DNA code do notresult in a change in the encoded protein. Further, this term isintended to encompass other minor variations in the sequence which maybe required to enhance expression in a particular system but in whichthe variations do not result in a decrease in biological activity of theencoded protein.

The term “functionally equivalent fragment/s” as used herein is intendedto refer to fragments of the GABA-B2 receptor that exhibit bindingspecificity and activity that is substantially equivalent to the GABA-B2receptor from which it/they is/are derived.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

Reference to percent homology made in this specification have beencalculated using the BLAST program blastn as described by Altschul, S.F. et al., “Capped BLAST and PSI-BLAST: a new generation of proteindatabase search programs”, Nucleic Acids Research, Vol. 25, No. 17, pp.3389-3402 (1997).

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

FIG. 1 provides the nucleotide sequence of a cDNA encoding the humanGABA-B2 receptor and includes the predicted amino acid sequence.

FIG. 2 shows the degree of identity between the predicted amino acidsequence of the human GABA-B2 and GABA-B1b receptors.

DETAILED DISCLOSURE OF THE INVENTION Human GABA-B2 Receptor cDNA

A human lippocampus cDNA library (Stratagene) was screened under lowstringency conditions with a 184 bp ³²P-labelled fragment (correspondingto nucleotides 2051 to 2235 of SEQ ID NO: 3) originated from a humanbrain EST clone (z43654). A cDNA clone encoding a complete human GABA-Bgene was obtained from the screen. The nucleotide sequence of the cDNAclone is shown as SEQ ID NO: 3 and encodes a protein of 941 amino acids(SEQ ID NO: 2) consisting of a large extracellular domain (amino acids 1to 480) followed by 7 hydrophobic regions (amino acids 481 to 494, 518to 545, 558 to 578, 597 to 618, 653 to 676, 691 to 713 and 719 to 743)typical of G-protein coupled receptors.

Sequence comparison with other G-protein coupled receptors identifyGABA-B1a, GABA-B1b (FIG. 1) and metabotropic glutamate receptors(Kaupinan, K. et al., Nature 386: 239-246, Mar. 20, 1997 and Pin, J. etal., Neuropharmacology 34: 1-26, 1995) as the most closely relatedgroup.

Northern analysis has identified brain as the tissue with the highestexpression of the human GABA-B2 mRNA. In particular, Northern analysisexperiments using multiple tissue Northern blots (Clontech) identifiedhigh levels of expression of the human GABA-B2 mRNA in the cerebellum,cerebral cortex, occipital pole, frontal lobe and temporal lobe. Lowerlevels of expression were seen in the thalamus, amygldala, hippocampus,substantia nigra, putamen, subthalamic nucleus, caudate nucleus, andmedulla. No apparent expression was seen in the spinal cord and corpuscallosum.

Further, in situ hybridisation of rat brain sections has identifieddiscrete areas of expression in the hippocampus, amygdala, the piriformcortex and also the hypothalamus. This mRNA distribution is consistentwith the expression of other subtypes of the GABA-B receptor family.

Chromosomal Localisation of Human GABA-B2 receptor gene

In order to determine the chromosomal localisation of the human GABA-B2receptor gene, the complete cDNA clone was nick-translated withbiotin-14-dAPT and hybridised in situ at a final concentration of 5ng/ml to metaphase chromosomal spreads from two normal males. Thefluoerescence in situ hybridisation (FISH) method was modified from thatpreviously described (Callen, DF et al., Ann Genet 33: 219-221 1990) inthat chromosomes were stained before analysis with both propidium iodide(as counterstain) and DAPI (for chromosome identification). Images ofmetaphase preparations were captured by a CCD camera and computerenhancement software.

Twenty metaphases from a first normal male were examined for fluorescentsignal. All of these metaphases showed signal on one or both chromatidsof chromosome 9 in the region 9q21. There was a total of 7 non-specificbackground dots observed in these 20 metaphases. A similar result wasobtained from hybridisation of the probe to 20 metaphases from a secondnormal male.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

4 1 10 PRT Homo sapiens 1 Met Ala Ser Pro Arg Ser Ser Gly Gln Pro 1 5 102 941 PRT Homo sapiens 2 Met Ala Ser Pro Arg Ser Ser Gly Gln Pro Gly ProPro Pro Pro Pro 1 5 10 15 Pro Pro Pro Pro Ala Arg Leu Leu Leu Leu LeuLeu Leu Pro Leu Leu 20 25 30 Leu Pro Leu Ala Pro Gly Ala Trp Gly Trp AlaArg Gly Ala Pro Arg 35 40 45 Pro Pro Pro Ser Ser Pro Pro Leu Ser Ile MetGly Leu Met Pro Leu 50 55 60 Thr Lys Glu Val Ala Lys Gly Ser Ile Gly ArgGly Val Leu Pro Ala 65 70 75 80 Val Glu Leu Ala Ile Glu Gln Ile Arg AsnGlu Ser Leu Leu Arg Pro 85 90 95 Tyr Phe Leu Asp Leu Arg Leu Tyr Asp ThrGlu Cys Asp Asn Ala Lys 100 105 110 Gly Leu Lys Ala Phe Tyr Asp Ala IleLys Tyr Gly Pro Asn His Leu 115 120 125 Met Val Phe Gly Gly Val Cys ProSer Val Thr Ser Ile Ile Ala Glu 130 135 140 Ser Leu Gln Gly Trp Asn LeuVal Gln Leu Ser Phe Ala Ala Thr Thr 145 150 155 160 Pro Val Leu Ala AspLys Lys Lys Tyr Pro Tyr Phe Phe Arg Thr Val 165 170 175 Pro Ser Asp AsnAla Val Asn Pro Ala Ile Leu Lys Leu Leu Lys His 180 185 190 Tyr Gln TrpLys Arg Val Gly Thr Leu Thr Gln Asp Val Gln Arg Phe 195 200 205 Ser GluVal Arg Asn Asp Leu Thr Gly Val Leu Tyr Gly Glu Asp Ile 210 215 220 GluIle Ser Asp Thr Glu Ser Phe Ser Asn Asp Pro Cys Thr Ser Val 225 230 235240 Lys Lys Leu Lys Gly Asn Asp Val Arg Ile Ile Leu Gly Gln Phe Asp 245250 255 Gln Asn Met Ala Ala Lys Val Phe Cys Cys Ala Tyr Glu Glu Asn Met260 265 270 Tyr Gly Ser Lys Tyr Gln Trp Ile Ile Pro Gly Trp Tyr Glu ProSer 275 280 285 Trp Trp Glu Gln Val His Thr Glu Ala Asn Ser Ser Arg CysLeu Arg 290 295 300 Lys Asn Leu Leu Ala Ala Met Glu Gly Tyr Ile Gly ValAsp Phe Glu 305 310 315 320 Pro Leu Ser Ser Lys Gln Ile Lys Thr Ile SerGly Lys Thr Pro Gln 325 330 335 Gln Tyr Glu Arg Glu Tyr Asn Asn Lys ArgSer Gly Val Gly Pro Ser 340 345 350 Lys Phe His Gly Tyr Ala Tyr Asp GlyIle Trp Val Ile Ala Lys Thr 355 360 365 Leu Gln Arg Ala Met Glu Thr LeuHis Ala Ser Ser Arg His Gln Arg 370 375 380 Ile Gln Asp Phe Asn Tyr ThrAsp His Thr Leu Gly Arg Ile Ile Leu 385 390 395 400 Asn Ala Met Asn GluThr Asn Phe Phe Gly Val Thr Gly Gln Val Val 405 410 415 Phe Arg Asn GlyGlu Arg Met Glu Thr Ile Lys Phe Thr Gln Phe Gln 420 425 430 Asp Ser ArgGlu Val Lys Val Gly Glu Tyr Asn Ala Val Ala Asp Thr 435 440 445 Leu GluIle Ile Asn Asp Thr Ile Arg Phe Gln Gly Ser Glu Pro Pro 450 455 460 LysAsp Lys Thr Ile Ile Leu Glu Gln Leu Arg Lys Ile Ser Leu Pro 465 470 475480 Leu Tyr Ser Ile Leu Ser Ala Leu Thr Ile Leu Gly Met Ile Met Ala 485490 495 Ser Ala Phe Leu Phe Phe Asn Ile Lys Asn Arg Asn Gln Lys Leu Ile500 505 510 Lys Met Ser Ser Pro Tyr Met Asn Asn Leu Ile Ile Leu Gly GlyMet 515 520 525 Leu Ser Tyr Ala Ser Ile Phe Leu Phe Gly Leu Asp Gly SerPhe Val 530 535 540 Ser Glu Lys Thr Phe Glu Thr Leu Cys Thr Val Arg ThrTrp Ile Leu 545 550 555 560 Thr Val Gly Tyr Thr Thr Ala Phe Gly Ala MetPhe Ala Lys Thr Trp 565 570 575 Arg Val His Ala Ile Phe Lys Asn Val LysMet Lys Lys Lys Ile Ile 580 585 590 Lys Asp Gln Lys Leu Leu Val Ile ValGly Gly Met Leu Leu Ile Asp 595 600 605 Leu Cys Ile Leu Ile Cys Trp GlnAla Val Asp Pro Leu Arg Arg Thr 610 615 620 Val Glu Lys Tyr Ser Met GluPro Asp Pro Ala Gly Arg Asp Ile Ser 625 630 635 640 Ile Arg Pro Leu LeuGlu His Cys Glu Asn Thr His Met Thr Ile Trp 645 650 655 Leu Gly Ile ValTyr Ala Tyr Lys Gly Leu Leu Met Leu Phe Gly Cys 660 665 670 Phe Leu AlaTrp Glu Thr Arg Asn Val Ser Ile Pro Ala Leu Asn Asp 675 680 685 Ser LysTyr Ile Gly Met Ser Val Tyr Asn Val Gly Ile Met Cys Ile 690 695 700 IleGly Ala Ala Val Ser Phe Leu Thr Arg Asp Gln Pro Asn Val Gln 705 710 715720 Phe Cys Ile Val Ala Leu Val Ile Ile Phe Cys Ser Thr Ile Thr Leu 725730 735 Cys Leu Val Phe Val Pro Lys Leu Ile Thr Leu Arg Thr Asn Pro Asp740 745 750 Ala Ala Thr Gln Asn Arg Arg Phe Gln Phe Thr Gln Asn Gln LysLys 755 760 765 Glu Asp Ser Lys Thr Ser Thr Ser Val Thr Ser Val Asn GlnAla Ser 770 775 780 Thr Ser Arg Leu Glu Gly Leu Gln Ser Glu Asn His ArgLeu Arg Met 785 790 795 800 Lys Ile Thr Glu Leu Asp Lys Asp Leu Glu GluVal Thr Met Gln Leu 805 810 815 Gln Asp Thr Pro Glu Lys Thr Thr Tyr IleLys Gln Asn His Tyr Gln 820 825 830 Glu Leu Asn Asp Ile Leu Asn Leu GlyAsn Phe Thr Glu Ser Thr Asp 835 840 845 Gly Gly Lys Ala Ile Leu Lys AsnHis Leu Asp Gln Asn Pro Gln Leu 850 855 860 Gln Trp Asn Thr Thr Glu ProSer Arg Thr Cys Lys Asp Pro Ile Glu 865 870 875 880 Asp Ile Asn Ser ProGlu His Ile Gln Arg Arg Leu Ser Leu Gln Leu 885 890 895 Pro Ile Leu HisHis Ala Tyr Leu Pro Ser Ile Gly Gly Val Asp Ala 900 905 910 Ser Cys ValSer Pro Cys Val Ser Pro Thr Ala Ser Pro Arg His Arg 915 920 925 His ValPro Pro Ser Phe Arg Val Met Val Ser Gly Leu 930 935 940 3 3256 DNA Homosapiens 3 gaattccgac gggcggtgtg tacaaagggc agggacttaa tcaacgcaagcttatgaccc 60 gcactccttg gcgcggggcg gcgggccggg ccaggccatg cgggccgagtgagccggcgc 120 ccgcagcccg cggcgcggca tggcttcccc gcggagctcc gggcagcccgggccgccgcc 180 gccgccgcca ccgccgcccg cgcgcctgct actgctactg ctgctgccgctgctgctgcc 240 tctggcgccc ggggcctggg gctgggcgcg gggcgccccc cggccgccgcccagcagccc 300 gccgctctcc atcatgggcc tcatgccgct caccaaggag gtggccaagggcagcatcgg 360 gcgcggtgtg ctccccgccg tggaactggc catcgagcag atccgcaacgagtcactcct 420 gcgcccctac ttcctcgacc tgcggctcta tgacacggag tgcgacaacgcaaaagggtt 480 gaaagccttc tacgatgcga taaaatacgg gccgaaccac ttgatggtgtttggaggcgt 540 ctgtccatcc gtcacatcca tcattgcaga gtccctccaa ggctggaatctggtgcagct 600 ttcttttgct gcaaccacgc ctgttctagc cgataagaaa aaatacccttatttctttcg 660 gaccgtccca tcagacaatg cggtgaatcc agccattctg aagttgctcaagcactacca 720 gtggaagcgc gtgggcacgc tgacgcaaga cgttcagagg ttctctgaggtgcggaatga 780 cctgactgga gttctgtatg gcgaggacat tgagatttca gacaccgagagcttctccaa 840 cgatccctgt accagtgtca aaaagctgaa ggggaatgat gtgcggatcatccttggcca 900 gtttgaccag aatatggcag caaaagtgtt ctgttgtgca tacgaggagaacatgtatgg 960 tagtaaatat cagtggatca ttccgggctg gtacgagcct tcttggtgggagcaggtgca 1020 cacggaagcc aactcatccc gctgcctccg gaagaatctg cttgctgccatggagggcta 1080 cattggcgtg gatttcgagc ccctgagctc caagcagatc aagaccatctcaggaaagac 1140 tccacagcag tatgagagag agtacaacaa caagcggtca ggcgtggggcccagcaagtt 1200 ccacgggtac gcctacgatg gcatctgggt catcgccaag acactgcagagggccatgga 1260 gacactgcat gccagcagcc ggcaccagcg gatccaggac ttcaactacacggaccacac 1320 gctgggcagg atcatcctca atgccatgaa cgagaccaac ttcttcggggtcacgggtca 1380 agttgtattc cggaatgggg agagaatgga gaccattaaa tttactcaatttcaagacag 1440 cagggaggtg aaggtgggag agtacaacgc tgtggccgac acactggagatcatcaatga 1500 caccatcagg ttccaagggt ccgaaccacc aaaagacaag accatcatcctggagcagct 1560 gcggaagatc tccctacctc tctacagcat cctctctgcc ctcaccatcctcgggatgat 1620 catggccagt gcttttctct tcttcaacat caagaaccgg aatcagaagctcataaagat 1680 gtcgagtcca tacatgaaca accttatcat ccttggaggg atgctctcctatgcttccat 1740 atttctcttt ggccttgatg gatcctttgt ctctgaaaag acctttgaaacactttgcac 1800 cgtcaggacc tggattctca ccgtgggcta cacgaccgct tttggggccatgtttgcaaa 1860 gacctggaga gtccacgcca tcttcaaaaa tgtgaaaatg aagaagaagatcatcaagga 1920 ccagaaactg cttgtgatcg tggggggcat gctgctgatc gacctgtgtatcctgatctg 1980 ctggcaggct gtggaccccc tgcgaaggac agtggagaag tacagcatggagccggaccc 2040 agcaggacgg gatatctcca tccgccctct cctggagcac tgtgagaacacccatatgac 2100 catctggctt ggcatcgtct atgcctacaa gggacttctc atgttgttcggttgtttctt 2160 agcttgggag acccgcaacg tcagcatccc cgcactcaac gacagcaagtacatcgggat 2220 gagtgtctac aacgtgggga tcatgtgcat catcggggcc gctgtctccttcctgacccg 2280 ggaccagccc aatgtgcagt tctgcatcgt ggctctggtc atcatcttctgcagcaccat 2340 caccctctgc ctggtattcg tgccgaagct catcaccctg agaacaaacccagatgcagc 2400 aacgcagaac aggcgattcc agttcactca gaatcagaag aaagaagattctaaaacgtc 2460 cacctcggtc accagtgtga accaagccag cacatcccgc ctggagggcctacagtcaga 2520 aaaccatcgc ctgcgaatga agatcacaga gctggataaa gacttggaagaggtcaccat 2580 gcagctgcag gacacaccag aaaagaccac ctacattaaa cagaaccactaccaagagct 2640 caatgacatc ctcaacctgg gaaacttcac tgagagcaca gatggaggaaaggccatttt 2700 aaaaaatcac ctcgatcaaa atccccagct acagtggaac acaacagagccctctcgaac 2760 atgcaaagat cctatagaag atataaactc tccagaacac atccagcgtcggctgtccct 2820 ccagctcccc atcctccacc acgcctacct cccatccatc ggaggcgtggacgccagctg 2880 tgtcagcccc tgcgtcagcc ccaccgccag cccccgccac agacatgtgccaccctcctt 2940 ccgagtcatg gtctcgggcc tgtaagggtg ggaggcctgg cccgggcctcccccgtgaca 3000 gaaccacact gggcagaggg gtctgctgca gaaacactgt cggctctggctgcggagaag 3060 ctgggcacca tggctggcct ctcaggacca ctcggatggc actcaggtggacaggacggg 3120 gcagggggag acttggcacc tgacctcgag ccttatttgt gaagtccttatttcttcaca 3180 aagaagagga acggaaatgg gacgtcttcc ttaacatctg caaacaaggaggcgctggga 3240 tatcaaactg gaattc 3256 4 844 PRT Homo sapiens 4 Met GlyPro Gly Gly Pro Cys Thr Pro Val Gly Trp Pro Leu Pro Leu 1 5 10 15 LeuLeu Val Met Ala Ala Gly Val Ala Pro Val Trp Ala Ser His Ser 20 25 30 ProHis Leu Pro Arg Pro His Pro Arg Val Pro Pro His Pro Ser Ser 35 40 45 GluArg Arg Ala Val Tyr Ile Gly Ala Leu Phe Pro Met Ser Gly Gly 50 55 60 TrpPro Gly Gly Gln Ala Cys Gln Pro Ala Val Glu Met Ala Leu Glu 65 70 75 80Asp Val Asn Ser Arg Arg Asp Ile Leu Pro Asp Tyr Glu Leu Lys Leu 85 90 95Ile His His Asp Ser Lys Cys Asp Pro Gly Gln Ala Thr Lys Tyr Leu 100 105110 Tyr Glu Leu Leu Tyr Asn Asp Pro Ile Lys Ile Ile Leu Met Pro Gly 115120 125 Cys Ser Ser Val Ser Thr Leu Val Ala Glu Ala Ala Arg Met Trp Asn130 135 140 Leu Ile Val Leu Ser Tyr Gly Ser Ser Ser Pro Ala Leu Ser AsnArg 145 150 155 160 Gln Arg Phe Pro Thr Phe Phe Arg Thr His Pro Ser AlaThr Leu His 165 170 175 Asn Pro Thr Arg Val Lys Leu Phe Glu Lys Trp GlyTrp Lys Lys Ile 180 185 190 Ala Thr Ile Gln Gln Thr Thr Glu Val Phe ThrSer Thr Leu Asp Asp 195 200 205 Leu Glu Glu Arg Val Lys Glu Ala Gly IleGlu Ile Thr Phe Arg Gln 210 215 220 Ser Phe Phe Ser Asp Pro Ala Val ProVal Lys Asn Leu Lys Arg Gln 225 230 235 240 Asp Ala Arg Ile Ile Val GlyLeu Phe Tyr Glu Thr Glu Ala Arg Lys 245 250 255 Val Phe Cys Glu Val TyrLys Glu Arg Leu Phe Gly Lys Lys Tyr Val 260 265 270 Trp Phe Leu Ile GlyTrp Tyr Ala Asp Asn Trp Phe Lys Ile Tyr Asp 275 280 285 Pro Ser Ile AsnCys Thr Val Asp Glu Met Thr Glu Ala Val Glu Gly 290 295 300 His Ile ThrThr Glu Ile Val Met Leu Asn Pro Ala Asn Thr Arg Ser 305 310 315 320 IleSer Asn Met Thr Ser Gln Glu Phe Val Glu Lys Leu Thr Lys Arg 325 330 335Leu Lys Arg His Pro Glu Glu Thr Gly Gly Phe Gln Glu Ala Pro Leu 340 345350 Ala Tyr Asp Ala Ile Trp Ala Leu Ala Leu Ala Leu Asn Lys Thr Ser 355360 365 Gly Gly Gly Gly Arg Ser Gly Val Arg Leu Glu Asp Phe Asn Tyr Asn370 375 380 Asn Gln Thr Ile Thr Asp Gln Ile Tyr Arg Ala Met Asn Ser SerSer 385 390 395 400 Phe Glu Gly Val Ser Gly His Val Val Phe Asp Ala SerGly Ser Arg 405 410 415 Met Ala Trp Thr Leu Ile Glu Gln Pro Gln Gly GlySer Tyr Lys Lys 420 425 430 Ile Gly Tyr Tyr Asp Ser Thr Lys Asp Asp LeuSer Trp Ser Lys Thr 435 440 445 Asp Lys Trp Ile Gly Gly Ser Pro Pro AlaAsp Gln Thr Leu Val Ile 450 455 460 Lys Thr Phe Arg Phe Leu Ser Gln LysLeu Phe Ile Ser Val Ser Val 465 470 475 480 Leu Ser Ser Leu Gly Ile ValLeu Ala Val Val Cys Leu Ser Phe Asn 485 490 495 Ile Tyr Asn Ser His ValArg Tyr Ile Gln Asn Ser Gln Pro Asn Leu 500 505 510 Asn Asn Leu Thr AlaVal Gly Cys Ser Leu Ala Leu Ala Ala Val Phe 515 520 525 Pro Leu Gly LeuAsp Gly Tyr His Ile Gly Arg Asn Gln Phe Pro Phe 530 535 540 Val Cys GlnAla Arg Leu Trp Leu Leu Gly Leu Gly Phe Ser Leu Gly 545 550 555 560 TyrGly Ser Met Phe Thr Lys Ile Trp Trp Val His Thr Gly Phe Thr 565 570 575Lys Lys Glu Glu Lys Lys Glu Trp Arg Lys Thr Leu Glu Pro Trp Lys 580 585590 Leu Tyr Ala Thr Val Gly Leu Leu Val Gly Met Asp Val Leu Thr Leu 595600 605 Ala Ile Trp Gln Ile Val Asp Pro Leu His Arg Thr Ile Glu Thr Phe610 615 620 Ala Lys Glu Glu Pro Lys Glu Asp Ile Asp Val Ser Ile Leu ProGln 625 630 635 640 Leu Glu His Cys Ser Ser Arg Lys Met Asn Thr Trp LeuGly Ile Phe 645 650 655 Tyr Gly Tyr Lys Gly Leu Leu Leu Leu Leu Gly IlePhe Leu Ala Tyr 660 665 670 Glu Thr Lys Ser Val Ser Thr Glu Lys Ile AsnAsp His Arg Ala Val 675 680 685 Gly Met Ala Ile Tyr Asn Val Ala Val LeuCys Leu Ile Thr Ala Pro 690 695 700 Val Thr Met Ile Leu Ser Ser Gln GlnAsp Ala Ala Phe Ala Phe Ala 705 710 715 720 Ser Leu Ala Ile Val Phe SerSer Tyr Ile Thr Leu Val Val Leu Phe 725 730 735 Val Pro Lys Met Arg ArgLeu Ile Thr Arg Gly Glu Trp Gln Ser Glu 740 745 750 Ala Gln Asp Thr MetLys Thr Gly Ser Ser Thr Asn Asn Asn Glu Glu 755 760 765 Glu Lys Ser ArgLeu Leu Glu Lys Glu Asn Arg Glu Leu Glu Lys Ile 770 775 780 Ile Ala GluLys Glu Glu Arg Val Ser Glu Leu Arg His Gln Leu Gln 785 790 795 800 SerArg Gln Gln Leu Arg Ser Arg Arg His Pro Pro Thr Pro Pro Glu 805 810 815Pro Ser Gly Gly Leu Pro Arg Gly Pro Pro Glu Pro Pro Asp Arg Leu 820 825830 Ser Cys Asp Gly Ser Arg Val His Leu Leu Tyr Lys 835 840

What is claimed is:
 1. An isolated polynucleotide molecule that encodesa GABA-B receptor polypeptide wherein said polynucleotide moleculecomprises a nucleotide sequence selected from the group consisting of:(i) the sequence set forth in SEQ ID NO; 3; (ii) the sequence consistingof nucieotides 140 to 2962 of SEQ ID NO: 3; and (iii) a sequence thatencodes the amino acid sequence set forth in SEQ ID NO:
 2. 2. Anisolated polynucleotide molecule according to claim 1, wherein thepolynucleotide molecule encodes a GABA-B receptor polypeptide of humanorigin of 941 amino acids in length.
 3. An isolated polynucleotidemolecule that encodes a human GABA-B receptor polypeptide having anamino add sequence corresponding to the sequence set forth in SEQ ID NO:2.
 4. An isolated polynucleotide molecule that encodes a GABA-B receptorpolypeptide, wherein said polynucleotide molecule comprises a nucleotidesequence consisting of nucleotides 1 to 3256 of SEQ ID NO:
 3. 5. Anisolated polynucleotide molecule that encodes a GABA-B receptorpolypeptide, wherein said polynucleotide molecule comprises a nucleotidesequence consisting of nucleotides 140 to 2962 of SEQ ID NO:
 3. 6. Aplasmid or expression vector comprising a polynucleotide moleculeaccording to claim
 1. 7. A host cell transformed with a polynucleotidemolecule according to claim
 1. 8. A host cell according to claim 7,wherein the cell is a mammalian or insect cell.
 9. A host cell accordingto claim 8, wherein the cell is a Chinese hamster ovary (CHO) cell,human embryonic kidney (HEK) 293 cell or an insect Sf9 cell.
 10. A hostcell according to claim 7, wherein the cell expresses on the cell'ssurface the GABA-B receptor polypeptide encoded by the polynucleotidetransformed into the cell.
 11. A method of producing a GABA-B receptorpolypeptide, said method comprising culturing a host cell transformedwith the polynucleotide of claim 1 under conditions sufficient forexpression of the GABA-B receptor polypeptide encoded by thepolynucleotide to occur.
 12. The method of claim 11 further comprisingrecovering the GABA-B receptor polypeptide from the host cellsubstantially free of other proteins.
 13. A method of producing a GABA-Breceptor polypeptide, said method comprising introducing thepolynucleotide of claim 1 encoding a GABA-B receptor polypeptide into acell thereby producing a transformed cell and then growing thetransformed cell under conditions sufficient for expression of theencoded GABA-B receptor polypeptide to occur.
 14. A GABA-B receptorpolypeptide comprising the amino acid sequence set forth In SEQ ID NO:2, substantially free of other human proteins.
 15. A receptorpolypeptide according to claim 14, wherein said polypeptide is a humanreceptor of 941 amino acids.
 16. A receptor polypeptide according toclaim 14, wherein said receptor has an amino acid sequence correspondingto that shown as SEQ ID NO: 2.